Date of Award

8-31-2014

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Manickam Sugumaran

Second Advisor

Jason Evans

Third Advisor

Steven Ackerman

Abstract

Hardening of cuticle, a vital process for the survival of all insects is achieved by the covalent crosslinking of cuticular proteins and chitin by reactive intermediates generated from sclerotizing precursors - N-acetyldopamine (NADA) and N-β-alanyldopamine (NBAD). It has been known that the cuticles using NADA are usually lighter in color, while those using NBAD are brown to black in color. In order to identify the biochemical reasons for this difference, several biochemical studies were conducted.

The catecholamine products present in the puparial cuticle of Sarcophaga bullata were isolated and analyzed by High Performance Liquid Chromatography (HPLC) and Mass Spectrometry. A set of tetramers of dehydro NADA was identified by liquid chromatography - mass spectrometry. Two other compounds - N-β-alanylnorepinephrine (NBANE) and N-β-alanylarterenone - were identified tentatively by HPLC and ultraviolet spectral analysis. These studies show that both NADA and NBAD are used for puparial cuticular hardening.

Sarcophagid quinone isomerase readily converted NBAD quinone to its quinone methide, which reacted rapidly with water forming the Michael-1,6-addition product, NBANE. Phenoloxidase generated NBANE quinone served as a substrate for quinone isomerase generating N-β-alanylarterenone. HPLC Mass spectral studies revealed the productions of NBANE, N-β-alanylarterenone, dehydro NBAD, and oligomers of dehydro NBAD in reaction mixture containing NBAD, phenoloxidase, quinone isomerase and quinone methide isomerase.

Tyrosinase oxidized dehydro NBAD to a transient quinone methide imine amide (QMIA) before producing oligomers of dehydro NBAD. Liquid chromatography mass spectrometry analysis of the reaction confirmed the production of dimers and other oligomers. Laccase also oxidized dehydro NBAD to oligomers, with out producing QMIA, probably by free radical coupling. Dehydro NBAD is sensitive to alkaline pH and exhibited rapid nonenzymatic oxidation. These results indicate that dehydro NBAD behaves much like dehydro NADA. The difference in color of cuticle using NBAD and NADA seems to come from the differences in reactivity of NBAD quinone, NBAD quinone methide, and NBAD QMIA and the way the enzymes utilize these reactive intermediates both temporal and spatial in the cuticle during sclerotization.

Comments

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